Various proteins, such as enzymes are used increasingly in industry and householding. Being proteins they will be able to stimulate an immunological response in man and animals.
Other proteins, such as hormones are used increasingly in medicine for the treatment and/or diagnosis of various conditions of illness and disease, whereby these proteins are injected into or otherwise presented to the immune system of animals, including man.
Depending on the way of presentation the stimulation can lead to production of various types of antibodies, and to a cellular response too. Of these routes at least one, being the one type of antibody can have adverse effect in man and animals. The production of IgE and maybe IgG4 can lead to an allergic state, giving symptoms like f.ex. rhinitis, conjunctivitis or other.
It cannot be excluded that other immunologically based adverse reactions will be seen with the increased use of these proteins.
These drawbacks in the use of proteins have been known for many years and various solutions have been used for solving these.
Within the field of industrial enzymes the most frequently used method for avoiding problems with allergic reactions from exposure to the products has been confectioning the enzymes in various ways by immobilizing, granulating and coating the enzymes thereby avoiding any release of the proteinaceous material during normal handling and storage.
However, this solution poses various problems in relation to bringing the enzyme into contact with the material with which it is meant to interact, such as bringing the enzyme into solution etc., and also some release of the enzyme may occur provoking an allergic reaction in subjects sensitive to an exposure.
Within the field of medicine a much used method has been to use proteins of especially human or corresponding animal origin or at least of the same primary structure as the human (or the animal in question) protein.
This has proven to be successful in many instances, but it is not always possible to establish the existence of an animal equivalent to the protein in question, or it has been found that certain modified proteins possess certain advantages over the native protein. In such instances the risk of provoking an allergic response in the subject receiving treatment or being diagnosed exists.
Consequently a need exists for developing proteins that provoke less or no allergic reactions, while still retaining their original activity to a degree where they still are functional and may be used according to their original intent.
Those parts of a protein molecule that are recognized and bound immunologically are called epitopes. For molecules in the range of f.ex. 30000 Daltons there might be as many 12 epitopes.
Epitopes are being bound by immunological cells and by antibodies. Some epitopes are more important than other, these are called major in contrast to minor epitopes.
It has been found that slight changes in the epitopes will affect the binding strength in these bindings (Walsh, B. J. and Howden, M. E. H. (1989): A method for the detection of IgE binding sequences of allergens based on a modification of epitope mapping, Journal of Immunological Methods, 121, 275–280; Geysen, H. M., Tainen, J. A., Rodda, S. J., Mason, T. J., Alexander, H., Getzoff, E. D. and Lerner, R. A. (1987): Chemistry of Antibody Binding to a Protein. Science. 135, 1184–90; Geysen, H. M., Mason, T. J. and Rodda, S. J. (1988): Cognitive Features of Continuous Antigenic Determinants. Journal of molecular recognition. 1, 32–41.
This may result in a reduced importance of such a changed epitope, maybe converting it from a major to a minor epitope, or the binding strength may even be decreased to the level of high reversibility, i.e. no efficient binding. This phenomenon may be called epitope loss.
The above investigations were all performed on synthesized peptides mimicking the epitopes in question and variants thereof in order to establish the relative importance of the amino acid residues in the epitope being investigated, and consequently these investigations do not prove any effects to the epitopes in their native environment as parts of the complete protein, where phenomena only found in the tertiary structure of the protein, such as folding or the establishment of salt bridges etc., are in function.